为毒素休克大鼠红细胞蛋白激酶C活性的变化

大鼠注射内毒紊后0.5h,红细胞浆蛋白激酶C活性明显降低,细胞膜蛋白激酶C活性显著增高。由正常红细胞浆提纯的蛋白激酶C在体外能增加红细胞膜蛋白区带1、区带2、区带4.2、区带4.5以及区带4.9的~(32)P参入量。结果提示,内毒素休克早期大鼠红细胞蛋白激酶C被激活;激活的蛋白激酶C引起红细胞膜骨架蛋白的磷酸化,进而可能影响红细胞变形性。     

阵发性睡眠性血红蛋白尿症补体不敏感红细胞囊泡化现象的观察

经蛇毒因子溶血法纯化阵发性睡眠性血红蛋白尿症补体不敏感红细胞，用过氧化氢、中性多形核白细胞和卵黄磷脂酰胆碱人工脂质体分别与之孵育，通过检测上清乙酰胆碱脂酶活性了解红细胞囊泡化释放情况。结果显示阵发性睡眠性血红蛋尿症红细胞用过氧化氢或中性多形核白细胞诱导后上清中乙酰胆碱脂酶活性与正常对照无明显差别（Ｐ＞０．０５）；而在卵黄磷脂酰胆碱人工脂质体诱导下，随孵育时间延长，可见明显囊泡释放，第４小时乙酰胆碱     

老化红细胞的分离和鉴定

根据本实验室的工作,老化红细胞表面可结合自身IgG的特点,建立了一种新的分离年轻及老化红细胞的方法。由于Protein A可与IgG有特异结合,用活化的Sepharose 6MB结合Protein A制成亲和层析柱,以此分离老化红细胞。同时对分离的红细胞作了谷胱甘肽还原酶、谷胱甘肽过氧化物酶、过氧化氨酶、6-磷酸脱氢酶的活性和变形性及吞噬情况的比较。     

舒张功能障碍性心力衰竭发病机制的研究——血小板Ca~(2+),红细胞Ca~(2+)及其膜Ca~(2+),Mg~(2+)-ATP酶的变化

作者测定了42例收缩功能正常或大致正常,舒张功能障碍性轻、中度(Ⅰ°与Ⅱ°)心衰患者血小板Ca~(2+)、红细胞Ca~(2+)及其膜Ca~(2+),Mg~(2+)-ATP酶活性。结果表明:心衰组血小板Ca~(2+)与红细胞Ca~(2+)含量升高,Ca~(2+),Mg~(2+)-ATP酶活性下降,与对照组比较,P<0.001,其巾Ⅰ°与Ⅱ°间P<0.05或<0.001;不同心脏病间,上述改变以高血压性心脏病最明显,其次为肥厚型心肌病与冠心病。提示:细胞内Ca~(2+)超负荷可能是舒张功能障碍的原因之一。     

CD47-SIRPα在人类红细胞老化与吞噬过程中的作用

探讨CD47-SIRPα在人类红细胞衰老与吞噬过程中的作用。采用Western blot检测体外4℃和37℃保存红细胞(RBC)及老化过程中红细胞膜上CD47的表达量;用单核细胞单层分析试验(monocyte monolayer assay,MMA)观察人THP-1单核细胞系对不同年龄段红细胞和CD47抗体F(ab’)2端封闭红细胞的吞噬情况及Western blot检测THP-1细胞上SIRPα磷酸化情况。结果发现,无论是在体内老化还是体外老化过程中,红细胞膜上CD47的表达量均出现显著下降趋势,随着保存时间的延长,CD47的表达量最大可下降82.64%(4℃保存情况下);THP-1细胞对不同年龄段红细胞的吞噬情况存在差异性,更易吞噬年老红细胞,吞噬率为35.32%,对年轻红细胞的吞噬率为13.72%(n=7,P<0.02);对新鲜红细胞的吞噬率为2.22%,对CD47抗体F(ab’)2端封闭红细胞的吞噬率为31.51%(n=7,P<0.02);吞噬不同年龄段红细胞后,THP-1细胞上磷酸化SIRPα量不同,与吞噬年轻红细胞相比,吞噬年老红细胞后,磷酸化SIRPα的量下降了57.12%。表明人类红细胞的老化与吞噬过程和CD47-SIRPα相互作用存在密切关系。     

哌唑嗪对原发性高血压病人红细胞Ca~(2+),Mg~(2+)-ATP酶和钙调素的影响

本实验观察了原发性高血压(EH)病人红细胞Ca~(2+)Mg~(2+)-ATP酶活性,红细胞和血浆钙调素(CaM)的变化及哌唑嗪对其影响。结果表明:EH病人红细胞基础和最大Ca~(2+),Mg~(2+)-ATP酶活性显著下降,并与血压呈显著负相关;红细胞CaM活性也显著降低并与最大Ca~(2+),Mg~(2+)-ATP酶活性呈显著正相关;经哌唑嗪治疗后,EH病人红细胞Ca~(2+),Mg~(2+)-ATP酶活性和CaM变化不显著,但血浆CaM有所降低。提示EH病人红细胞钙代谢异常与外周阻力增高有密切关系,哌唑嗪对细胞和体液CaM有不同影响。     

川芎嗪对自发性高血压大鼠红细胞膜外翻囊泡钙摄取的影响

细胞膜Ca~(2+)转运功能异常在自发性高血压大鼠(SHR)发病过程中有重要作用。川芎嗪作为临床上广泛使用的活血化瘀药,其扩血管效应是否通过影响细胞膜Ca~(2+)转运机制,目前所知甚少。本文观察了川芎嗪对SHR和WKY大鼠红细胞膜外翻囊泡(IOV)Ca~(2+)摄取的影响,拟探讨在正常血压和高血压状态下其作用的差异和意义。     

老化红细胞研究的进展

各种动物每个细胞都有一定存活期,早在1946年Shemin及Rittenberg用~(14)C-甘氨酸测定红细胞的寿命,他们发现红细胞有规律的存活120天。相继Berlin等人测定了兔、猫及狗的红细胞寿命,也有同样现象,兔及猫红细胞存活20,狗100天。但是老化红细胞有什么特点?为什么120天即会消亡? 老化红细胞被吞噬细胞吞噬,这个现象虽早己发现,但吞噬的原因仍不清。近十几年来由于分子生物学的发展,生物技术的不断进步,对老化红细胞的研究有了不少的进展,现仅就以上一些问题作简略介绍。     

抗高血压因子对大鼠血管平滑肌钙内流的影响

实验观察了从原发性高血压病患者红细胞中提取的抗高血压因子(AHF)对自发性高血压大鼠(SHR)、肾性高血压大鼠(RHR)和正常血压的WKY大鼠和Wistar大鼠主动脉和肠系膜动咏平滑肌Ca~(2+)内流的影响。结果表明,SHR和RHR的肠系膜动脉Ca~(2+)内流显著高于主动脉;AHF可显著抑制SHR和RHR主动脉和肠系膜动脉Ca~(2+)内流,抑制作用呈剂量依赖性,且对肠系腆动脉Ca~(2+)内流抑制作用更明显;AHF也可抑制正常动物血管平滑肌Ca~(2+)内流。本工作提示,AHF的降压机制可能与其抑制血管平滑肌特别是小动脉血管平滑肌Ca~(2+)内流有关。     

钙调蛋白在免疫应答反应中的生物学效应及其表现

Ca~(2+)参与免疫应答反应的诱导和调节。从淋巴细胞活化到抗体合成以及非特异性炎症反应,各个阶段都少不了Ca~(2+)的作用。人们认为Ca~(2+)是环核苷酸诱导控制免疫应答反应的重要一环,但是对于二者之间的关系仍无定见。钙调蛋白(Calmodulin,CaM)的发现使我们对Ca~(2+)和环核苷酸调节免疫应答反应有了较为完整的概念。CaM是细胞内Ca~(2+)的重要受体,它与Ca~(2+)结合后发生构象改变而激活。同时又     

The effects of pentoxifylline on rat erythrocytes of different age

Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug-induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca2+; (2) membrane protein phosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5-2.5 mM) reduced intracellular Ca2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0-5.0 mM) increased Ca2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inhibition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca2+ entry into the cell.     

Erythrocyte ion transport and membrane potential in patients with chronic kidney failure

The membrane potential (delta E), the K+ concentration and the Ca2+-dependent potassium permeability of erythrocytes were studied in patients suffering from chronic renal insufficiency (CRI) in the terminal stage and undergoing hemodialysis. Increased absolute delta E value and reduced K+ concentration in the erythrocytes were revealed during the pathological condition. When erythrocytes were de-energized by NaF, addition of Ca2+ led to K+ elimination and proton rebound due to calcium-hydrogen exchange through the membrane. In some cases repeated injections of Ca2+ caused a series of rebounds, potassium penetration diminished in such instances. The proton rebound was absent in almost half of the patients with chronic renal insufficiency and the potassium flow was inhibited. The mechanisms of delta E increase are discussed and it is suggested that the inhibition of Ca2+-induced potassium penetration is due to structural changes of the erythrocyte membranes in CRI.     

Enhanced Ca2+ uptake by mouse erythrocytes in malarial (Plasmodium berghei) infection

Erythrocytes from Plasmodium berghei-infected mice on incubation either in plasma or artificial isotonic media showed an increase in uptake of 45Ca2+ compared with erythrocytes from uninfected mice. Infected cells (55% parasitaemia) incubated in plasma from normal or infected mice gave uptake rates of 9.8 and 8.1 nmol h-1 per 10(10) cells, assuming equilibrium between added 45Ca2+ and plasma Ca2+. Uptake rates of erythrocytes from infected mice were increased in the presence of glucose, with a rate of 15.0 nmol h-1 per 10(10) cells (52-58% parasitaemia) at 5 mM glucose, compared with 1.5 nmol h-1 per 10(10) cells in the absence of glucose. The enhancement of 45Ca2+ uptake was more pronounced with increasing parasitaemia, and in the fraction relatively enriched with erythrocytes carrying mature parasites. It is likely, therefore, that the enhancement is due to changes in membrane permeability accompanying parasite development. Enhanced haemolysis accompanied 45Ca2+ uptake of erythrocytes carrying mature parasites, but not of those carrying young parasites or uninfected erythrocytes. The possible role of an altered Ca2+ status in erythrocyte pathophysiology during malarial infection is discussed.     

血瘀证大鼠红细胞损伤模型的研制与应用

目的 :探索建立一种被动吸烟致大鼠以红细胞损伤为特点的血瘀证动物模型。方法 :通过SDS聚丙烯酰胺凝胶电泳、荧光偏振技术、细胞介电测量系统、扫描电镜、透射电镜、化学发光法、免疫花环法、比色法等对对照组、模型组、活血祛瘀治疗组的红细胞形态、结构、功能的多层次多指标进行测量。结果 :模型组红细胞在膜化学组分、膜巯基、膜脂流动性、膜电荷、红细胞膜ATPase活性、红细胞形态及超微结构、红细胞免疫受体及膜脂质过氧化酶等变化均较对照组明显异常 ,治疗组上述指标均接近对照组。结论 :该模型可作为血瘀证模型 ,尤以红细胞损伤为特点 ,不仅可用作血瘀证、活血化瘀法的研究 ,也可用以防治红细胞损伤药物的筛选 ,值得推广     

Calcium influx: a possible role for insulin modulation of intracellular distribution and activity of 6-phosphofructo-1-kinase in human erythrocytes

Human erythrocyte cells contain specific, active insulin receptor. However, the physiological relevance of this receptor is unclear. Here we show that Ca2+ influx is 4-fold higher in erythrocytes upon insulin stimulation. These effects are dose-dependent and are diminished by insulin concentrations of 150 nM and higher. The insulin-stimulated Ca2+ influx depends on a tyrosine-kinase activity and involves the verapamil-dependent Ca2+ channels. Elevated intracellular Ca2+, in association with the Ca2+-binding protein, calmodulin, stimulates erythrocytes 6-phosphofructo-1-kinase activity. This activation involves the detachment of the enzyme from erythrocyte membranes, which has been described as an important mechanism of glycolysis regulation on these cells. Altogether, these results support evidence that insulin may increases glucose consumption in human erythrocytes, through a mechanism involving Ca2+ influx, calmodulin and the detachment of 6-phosphofructo-1-kinase from the erythrocyte membrane.     

Suicidal erythrocyte death in sepsis

Sequelae of sepsis include anemia which presumably results from accelerated clearance of erythrocytes from circulating blood. The underlying mechanisms, however, remained hitherto elusive. Most recent studies disclosed that increased cytosolic Ca2+ activity and ceramide both trigger suicidal erythrocyte death (i.e., eryptosis), which is characterized by lipid scrambling of the cell membrane leading to phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing erythrocytes may adhere to vascular walls or may be engulfed by macrophages equipped with phosphatidylserine receptors. To explore whether sepsis leads to eryptosis, erythrocytes from healthy volunteers were exposed to plasma of patients suffering from sepsis, or to supernatants from sepsis producing pathogens. Then, phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Challenge of erythrocytes with plasma from the patients but not with plasma from healthy individuals triggered annexin V binding. The effect of patient plasma on erythrocyte annexin V binding was paralleled by formation of ceramide and a significant increase of cytosolic Ca2+ activity. Exposure of erythrocytes to supernatant of pathogens similarly induced eryptosis, an effect correlating with sphingomyelinase activity. The present observations disclose a novel pathophysiological mechanism leading to anemia and derangement of microcirculation during sepsis. Exposure to plasma from septic patients triggers phosphatidylserine exposure leading to adherence to the vascular wall and clearance from circulating blood.     

老化红细胞变形能力与膜钙依赖中性蛋白酶活性的关系

研究表明,老化红细胞变形能力明显降低,且其降低与血红蛋白浓度增高及膜弹性降低有关［１］。红细胞膜钙依赖中性蛋白酶（Ｃａｌｐａｉｎ）和它的内源性抑制剂（Ｃａｌｐａｓｔａｔｉｎ）形成红细胞中一个蛋白水解系统,参与红细胞中的信号传导,调节细胞形状、体积和细胞膜通透性,与高血压、细胞老化等生理、病理现象密切相关。Ｃａｌｐａｉｎ可限制性水解红细胞膜骨架蛋白和其它膜内蛋白,导致红细胞损伤［２,３］。而老化红细胞变形能力降低与Ｃａｌｐａｉｎ的关系尚不清楚,为此我们检测了４２例健康人老化及年轻红细胞变形能力、Ｃ…     

Verapamil prevents impairment in filterability of human erythrocytes exposed to oxidative stress

Effects of oxidative stress on intact human erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Exposure of erythrocytes to tBHP caused a marked decrease in filterability in a time-dependent manner. Erythrocytes exposed to tBHP also show an increase in mean corpuscular volume and a remarkable formation of methemoglobin (met-Hb) without any appearance of hemichromes that form Heinz bodies. High performance liquid chromatography demonstrated that the tBHP-treated erythrocytes exhibited an apparent decrease in the membrane phospholipid, phosphatidylethanolamine (PE). The decrease in PE was inhibited by pretreatment with ascorbate, but not with verapamil. SDS-polyacrylamide gel electrophoresis of the tBHP-treated erythrocyte membrane showed a degradation of spectrin, band 3, band 4.2, and band 4.5, accompanied by the appearance of low-molecular-weight products. The degradation of the membrane proteins was not prevented by pretreatment with verapamil or ascorbate. However, the pretreatment with verapamil but not with ascorbate revealed significant inhibition of the tBHP-induced impairment in filterability in the presence of extracellular Ca2+. Thus, the present study shows that verapamil, a potent drug in reperfusion therapy, plays an important role in protection against oxidative injury, based on a close linkage among decreased filterability, met-Hb formation, and impaired membrane integrity.     

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates with the erythrocyte membrane skeletal protein, band 4.1

Several proteins synthesized by mature asexual stages of Plasmodium falciparum interact with the erythrocyte membrane skeleton. One of these is the mature-parasite-infected erythrocyte surface antigen (MESA; also called PfEMP2), a phosphoprotein of 250-300 kDa, which is found on the internal face of the erythrocyte membrane. When MESA is precipitated with anti-MESA antibodies, another phosphoprotein of 80 kDa is co-precipitated. This 80-kDa phosphoprotein was identified by peptide mapping as the erythrocyte membrane component band 4.1. Thus, MESA is apparently anchored at the erythrocyte membrane through an association with band 4.1. Band 4.1 is more intensely phosphorylated in infected erythrocytes and is increased in relative molecular mass in erythrocytes infected by isolates of P. falciparum that cytoadhere.     

Band 3 clustering promotes the exposure of neoantigens in Plasmodium falciparum-infected erythrocytes

Erythrocytes infected with the human malaria parasite Plasmodium falciparum become structurally and antigenically modified as a consequence of intracellular parasite development. The new antigens that appear on the surface of the infected erythrocyte originate from parasite-encoded proteins and by modification of the erythrocyte membrane protein band 3. Here, we show that anti-peptide antibodies generated against an amino acid sequence (YETFSKLIKIFQDH) of human band 3, and previously identified as mediating adhesion of infected erythrocytes to CD36, recognized P. falciparum-infected erythrocytes. In addition, sera from individuals living in a malaria endemic area (and who are presumably immune) contained immunoglobulins specific for this region of band 3. The anti-peptide antibodies reacted with the surface excrescences (knobs) on falciparum-infected erythrocytes. In uninfected erythrocytes, the band 3 region was cryptic and its exposure on the falciparum-infected erythrocyte surface required clustering of band 3 protein. Thus, a parasite-induced modification of band 3 promotes adhesion and induces antigenic changes in the P. falciparum-infected erythrocyte.